The long-term objective of this research is to understand the mechanisms that enable Epstein-Barr virus (EBV) to persist in a latent state within its human host and thus contribute to the development of EBV-associated tumors and lymphoproliferative disease. A fundamental prerequisite to fulfilling this objective is defining the regulatory pathways that control expression of the EBV EBNA-1 protein, which is essential for the maintenance of a latent EBV infection. EBNA-1 expression within several tumors and in latently-infected lymphocytes of health individuals is mediated by the promoter Qp. EBNA-1 is also expressed upon induction of the virus replication cycle via the Fp promoter, which overlaps Qp in the EBV genome. We propose the following aims to reach our long-term objective: 1) Elucidate the mechanism of Qp activation through the positive cis-regulatory elements DRE-1 and DRE-2 identified in the initial grant period; 2) Define the regulation of EBNA-1 expression by proteins that bind the Region III EBNA-1 binding sites adjacent to Qp; and 3) Elucidate the biological significance of EBNA-1 expression during EBV replication. Under Aim 1, we will delineate the key DNA-protein and protein-protein interactions that activate EBNA-1 expression through the DRE-1 and DRE-2 regulatory elements of Qp. This will include the identification, and if needed the purification and cDNA cloning, of the cellular proteins that act in trans with these elements. Under Aim 2 we will determine the relative roles that EBNA-1 and E2F contribute to regulation of QP in trans with the Region III EBNA-1 binding domain within the context of the cell cycle. These studies will be integrated with Aim 1 to determine how EBNA-1 and E2F and its associated factors ultimately regulate Qp. To elucidate the function and biological significance of EBNA-1 expression during the EBV replication cycle (Aim 3) we will determine: i) whether EBNA-1 is a virion component; and if so ii) whether virion-associated EBNA-1 enhances EBV transformation efficiency through early gene- regulatory events; and iii) whether EBNA-1 performs an essential function during replication.